DELETED IMMUNOGLOBULINS
Volume, Ithaca, N. Y., Cornel1 University Press, in press. Laties, G. G., and Treffry, T. (1969), Tissue Cell I, 575. Lehninger, A. L. (1970), Biochemistry, New York, N. Y., Worth Publishing Co. Meijer, A. J., Tager, J. M., and Van Dam, K. (1969), in The Energy Level and Metabolic Control in Mitochondria, Papa, S., Tager, J. M., Quagliariello, E., and Slater, E. C., Ed., Bari, Adriatica Editrice. Mitchell, P. (1966), in Regulation of Metabolic Processes in Mitochondria, Tager, J. M., Papa, S., Quagliariello, E., and Slater, E. C., Ed., New York, N. Y., Elsevier, p 65.
Raison, J. K., Laties, G. G., and Crompton, M. (1973b), J.Bioenerg. (in press). Raison, J. K., and Lyons, J. M. (1971), Proc. Nut. Acad. Sci. U. S. 68,2092. Raison, J. K., Lyons, J. M., and Campbell, L. C. (1973a), J . Bioenerg. 4 (in press). Ribereau-Gayon, G., and Laties, G. G. (1969), C. R. Acad. Sei., Ser. A 268,2290. VandenBergh, S. G. (1964), Bi0chem.J. 93,128. Wiskich, J. T., and Bonner, W. D., Jr. (1963), Plant Physiol. 38, 594.
A Technique for the Detection of Deleted Immunoglobulin Heavy Chains? Blas Frangionex
A method, “carboxymethylcysteine diagonal map,” has been developed to detect immunoglobulins with structural defects. It is based on the comparison of all cysteine-containing peptides present in the molecule with their normal counterparts. Immunoglobulin molecules have a high content of
ABSTRACT:
A
mino acid sequence studies of several y heavy-chain disease (yHCD)l proteins (Frangione and Milstein, 1969; Franklin and Frangione, 1971; Cooper et al., 1972) and the heavy (H) chain of a myeloma protein (Fett et al., 1973) have shown internal deletions ranging in size from 15 to 240 residues. These results have indicated (1) that these smaller synthetic products resulted from abnormalities of gene expression rather than degradation of intact molecules and (2) that codons GAA-GAG specifying glutamic acid at position 216 (yl numbering) (Edelman et al., 1969) have special significance since in three instances reinitiation of normal synthesis after an internal deletion started at position 216 (Frangione and Milstein, 1969; Franklin and Frangione, 1971; Cooper et al., 1972) and in one case the gap commenced at the same position (Fett et af., 1973). Since the interpretation of these data is not yet clear and since studies of such proteins promise to be of great value in developing theories to explain the genetic mechanisms involved in the control of immunoglobulin synthesis, a method has been developed to detect deleted immunoglobulins. It is based on the comparison of all cysteinecontaining peptides in the molecule with those in its normal counterpart. The method used employs “carboxymethyl~~~
t From
the New York University Medical Center, New York, New York 10016. Received April 2, 1973. This work was supported by the U. S. Public Health Service, National Institutes of Health Grants AM 01431, AM 02594, and AM 05064. $ Senior Investigator of the Arthritis Foundation, Inc. Present Address : Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, WC2, England. 1 Nomenclature of immunoglobulin and their chains follow the recommendation of the World Health Organization (W.H.O. Bull. 33, 721 (1965); 35, 953 (1966); 38, 151 (1968); 41, 975 (1969)). Myeloma proteins are designated by the first three letters of the patient’s name.
cysteine residues which are strategically spread along the chains and can be used as markers for different domains and interdomain regions. Since they occupy a distinct position on the map, the absence of one or more peptides defines rather accurately the nature of the defect.
cysteine diagonal maps” and differs from classical diagonal maps (Brown and Hartley, 1966) in that the proteins are subjected to mild thiol reduction and labeling with iodo[14C]acetic acid prior to the performance of the diagonal map. This results in the selective labeling of the labile interchain disulfide bridges. Materials and Methods
As previously described, 5 mg of purified protein (Frangione et a/., 1969a) was dissolved in 0.5 ml of 0.27 M Tris-HC1 buffer (pH 8.2) and mildly reduced with 0.005 M dithiothreitol at room temperature under N2. After incubation for 60 min at 37”, reduction was terminated by the addition of iodo[14C]acetic acid (0.01 M, specific activity 0.7 Ci/mol) and incubated at room temperature for another 60 min. The solution was then dialyzed overnight in 5 formic acid and digested with pepsin (Worthington, twice crystallized), enzyme-substrate ratio 1 :50 (w/w) for 14 hr at 37”, freeze-dried, dissolved in 0.2 M ammonium bicarbonate (pH 8.3), and digested with L(1-tosylamido-2-pheny1)ethyl chloromethyl ketone trypsin (Worthington), enzyme-substrate ratio 1 :50 (w/w) for 6 hr at 37”. The digest was dried, dissolved in 0.1 ml of water, and applied as a 3-cm band on 3MM Whatman paper and subjected to high-voltage paper electrophoresis in solvent-cooled tanks (pyridine-acetic acid-water, 1 :10 :190, v/v, pH 3.5) for 1 hr, 60 V/cm. A mixture of aspartic acid, glutamic acid, a-Dnp-lysine, and glycylalanine was applied at the sides of the paper as markers. Peptides containing S-carboxymethylcysteine were detected by autoradiography using Kodak Royal Blue Medical X-Ray film. The strip from this ionogram (without the markers) was incubated for 2 hr at room temperature in a desiccator conBIOCHEMISTRY,
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1 : "Carboxymethylcystine diagonal electrophoretic maps'' of partially reduced and labeled peptic-tryptic digests of (a) IgG k myeloma protein Car and (b) yl HCD protein Cra. Electrophoresis at pH 3.5 was carried out horizontally before oxidation and vertically after oxidation. At the botton autoradiographs after the first dimension. S-Carboxymethykystine sulfone containing peptides lying off the diagonal are hatched. Major cysteic acid ninhydrin positive peptides lying off the diagonal and corresponding to con, C K ~are ) circled. VK: stant Hchain domains (CHI, C N ~and H-chain, variable domain. Group of peptides lo the left of Cm3 belong to L chain: CL + VI. domains. H-L: heavy-light: H-H: heavy-heavy; L: light. Hinge or interdomain: region containing the H-H disulfide bonds located between CHI and Ca2 domains. Arrows indicate absence ofcorrespondingH chain domains.
FIGURE
taining a freshly prepared mixture of 50 ml of 98% (v/v) formic acid and 2.5 ml of 30% (v/v) H202in a Petri dish. (A convenient support for the strip IS a spiral glass rod.) The oxidized strip was removed from the desiccator and left for 1 hr in a fume cupboard to allow evaporation of the formic and performic acid. It was then stitched with a sewing machine to another sheet of paper and subjected to high-voltage electrophoresis under the original conditions but at right angles to the original direction. Peptide maps were stained with cadmium-ninhydrin; this solution contained 14% (v/v) cadmium acetate solution (6 g of cadmium acetate in 30% (v/v) acetic acid) in 1% (w/v) ninhydrin in acetone. S-Carboxymethylcysteine sulfone peptides were detected by autoradiography. The map can be used also as a guide to the selective purification of each cysteic acid and S-carboxymethylcysteine sulfone peptide to permit further characterization. For this purpose, 50 mg of protein is required. The initial map is used then to identify selected sections containing cysteine peptide. Purification of peptides, amino acid analysis, Edman degradation and N-terminal analysis were the same as published (Wolfenstein etal., 1971). Results To illustrate the method, the maps of four proteins, two belonging to the y l heavy-chain subclass and the other two to the 74 subclass, are presented. Emphasis will be given to the heavy chains of immunoglobulin G, although this method can be used for light chains, different subclasses of y chains, as well as a and p HCD proteins (unpublished observations). Figure 1 shows carboxymethylcysteine diagonal maps of (a) IgGlk myeloma protein Car and (b) yl HCD protein Cra. At the bottom of Figure 1 are the autoradiographs of partially reduced, labeled, and digested proteins obtained after firstdimensional electrophoresisat pH 3.5. Three radioactive bands (one very weak) containing S-carboxymethyl peptides are present in protein Car and two in protein Cra. Under the conditions used only interchain bridges were reduced and
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"Carboxymethylcystine diagonal electrophoretic maps'' of partially reduced and labeled peptic-tryptic digest of (a) l g A 4 k myeloma protein Wur and (b) y4 HCD protein Hal. Same conditions as in Figure 1. Notice thecompletelack oflabeled peptides in protein Hal (absence of inkrchain bonds). Arrows indicate absence of CHI and VH domain peptides. Cystine peptide corresponding to Cnl in protein Wur is more anodic than the homologous one in protein Car (compare Figures la and 2a) due to a charged amino acid replacement: Asp for Asn (Tables I and 11). Protein Wur shows cysteic acid peptides on the left of Cx3 and in the region between Cxl and Cn2 which corresponds to the L chain (unpublished observations). F~GURE 2:
labeled (Frangione et ai.,1969a), and intrachain bonds remain intact, After performic acid oxidation and diagonal electrophoretic separation at pH 3.5, intrachain bonds have been converted into a pair of cysteic acid peptides, each with an extra negative charge. Consequently, they will lie off the diagonal but vertically below each other, thus allowing the identification of each pair of cysteic acid peptides which was originally disulfide bridged. In addition, S-carboxymethylcysteine peptides will also lie off the diagonal since the pKa of the S-carboxymethyl group is lowered when it is oxidized to the sulfone form. Table I shows the amino acid sequence, function and position of S-carboxymethylcysteine peptides and cysteic acid peptides obtained from proteins Car and Cra. It can be seen that the intrachain S-S bond present in each domain (Edelman et al., 1969) or pseudosubunit (Frangione et ai., 1969b1, especially those belonging to the constant region ( C I I ~C112, , Cn3),run in a specificposition not only in the first but also in the second dimension. Thus the bridge corresponding to CHI runs ahead of the dipeptide glycylalanine in the first dimension and splits in two cysteic acid peptides after oxidation (la and Ib) and a low yield peptide (IC) which is related to l b (see Table I). The cystine peptide. present in Cn2 domain runs with glycylalanine in the first dimension and gives two peptides after the second dimension (2a and b, Table I). The one corresponding to CH3 domain runs between glycylalanine and e-Dnp-lysine and after oxidation gives a number of cysteic acid peptides (3a, b, and c) running between glutamic and aspartic acid after the second dimension. The cystine bridge present in the VH region of protein Car runs between Cn3 and C H (see ~ Figure I). It was also present in the diagonal map of the separated H chain (not shown). A group of cysteic acid peptides near to those present in Cn3 belongs to the L chain (k type) and will be discussed elsewhere. As expected three S-carboxymethyl sulfone peptides were obtained. Two of these are present on the H chain (H-H and H-L) and one (L) comes from the carboxy end of the k chain (see Table I and Figure 1). In the case of protein Cra only intrachain bonds coming from Cn2 and Cn3 domains were present as well as those
DELETED IMMUNOGLOBULINS
TABLE I : Sequence of Peptic-Tryptic Cysteine-Containing Peptides Obtained from IgG 1k Myeloma Protein Car and yl HeavyChain Disease (HCD) Protein Cra.'
Peptide Cms
Sequence CYa la lb IC
Domain
Protein CAR 200 Tyr-Ile-Cys-Asn-Val-Asn-His-Lys-Pro-Ser-Asn-Thr
I
Leu-GI y-Cys-Leu 144 Gly-Cy+Leu 321 cys-Lys
2a
I
2b
Thr-Pro-Glu-Val-Thr-Cys-Val-Val
3a
261 425 Phe-Ser- Cys-Ser-Val-Met
3b 3c
H-H H-L L
I
Leu-Thr-Cy +Leu 367 Thr-Cys-Leu 226 229 Thr-His-Thr-Cy s-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu 220 Ser-Cys-Asp-Lys J 214 Gly-Glu-Cys
Hinge (interdomain)
CL
Protein CRA c ys-Lys
2a
I I
Thr-Pro-Glu-Val-Thr-Cys-Val-Val
2b 3a
Phe-Ser- Cys-Ser-Val-Met
3b 3c
Leu-Thr-Cy s-Leu Thr-Cys-Leu
I H-H H-H~
CH 3
I
Thr-His-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu Ser-Cys-Asp-Lys
Hinge
a Partial or complete sequence of some peptides have been published (Frangione er af.,1969b; Franklin and Frangione, 1971). Numbering of protein Car was taken from proteins Eu (Edelman et al., 1969); Cms: S-carboxymethylcysteine sulfone peptides; Cya: cysteic acid peptides. Extra interheavy chain bond (see text).
involved in interchain binding (Table I and Figure 1). This molecule did not contain L chain (lack of peptide L) although it did have the H-L peptide which was shown to form a third interheavy chain bond (Franklin and Frangione, 1971). These findings are consistent with the detailed chemical studies, which proved an internal deletion of about 200 residues including the loops corresponding to VH and CHI (Fd fragment) (Franklin and Frangione, 1971). Figure 2 shows carboxymethylcysteine diagonal maps of (a) IgG 4k myeloma protein Wur and (b) 74 HCD protein Hal. Autoradiographs disclosed three radioactive bands in protein Wur and none in protein Hal, thus showing that HCD protein Hal lacked interchain bridges. As a result it existed as a monomer and could not be typed chemically (Frangione et af.,1969a). It could be placed in the 74 subclass by studying the carboxyl end of the molecule (Frangione et al., 1973), which is characteristic for the 7 4 subclass (Prahl, 1967). Performic acid diagonal electrophoresis maps disclosed only the presence of cysteine bridges present in cH2 and cH3.
It was concluded that protein Hal lacks not only the loops present in VH and CHI but also the regions responsible for interchain binding. Amino acid sequence studies (Frangione et al., 1973) indicated that it contained a gap of about 240 residues, starting 10 residues from the N-terminal end, and that normal sequence apparently resumed beyond the hinge at a methionine residue (position 252). The carboxymethylcysteine diagonal map of protein Wur (Figure 2) showed cysteic acid peptides coming from VH, CHI, cH2, and cH3 domains (those from L chain are not discussed here) and three S-carboxymethylcysteine sulfone peptides: H-H, H-L, and L. The H-L peptide was ninhydrin negative since proline is the N-terminal residue (Table 11). The amino acid sequence of cysteine-containing peptides and function of proteins Wur and Hal, are shown in Table 11. Note that the cystine peptide present in CHI of protein Wur is more anodic than the homologous one present in protein Car (Figure 1). This is due to an amino acid substitution in peptides l a (Asp, instead of Asn, Tables I and 11). BIOCHEMISTRY,
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Peptic-Tryptic Cysteine-Containing Peptides Obtained from IgG 4k Myeloma Protein Wur and 94 HeavyChain Disease (HCD) Protein Hal.’
TABLE 11: Sequence of
Sequence
Peptide
Domain
Protein WUR Cms
CYa la
Tyr-Thr- Cys-Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr
I
lb IC 2a
Leu-Gly-Cys-Leu Gly-Cys-Leu
2b 3a
Thr-Pro-Glu-Val-Thr-Cys-Val-Val
3b 3c
CH 1 cys-Lys
I
cH2
Phe-Ser- Cys-Ser-Val-Met
I
Leu-Thr-Cys-Leu Thr-Cys-Leu
CH 3
H-H
Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Ser-Glu-PheHinge
H-L L
Pro-Leu- Ala-Pro-Cy s-Ser-Arg Gly -Glu-Cys
(interdomain) CH 1 CL
Protein HAL cys-Lys
-
2a 2b 3a
Thr -Pro-Glu-Val-Thr-Cys-Val-Val Phe-Ser- Cys-Ser-Val-Met
3b
Leu-Thr-Cys-Leu
3c
Thr-Cys-Leu
1
I
‘Partial sequence of H-H peptide from protein Wur was previously reported (Frangione et al., 1969b). Italics indicates amino acid substitutions in homologous regions between y4 and y l chains. Discussion During studies of the interchain disulfide bonds of myeloma proteins (Frangione et al., 1969b, 1971; Frangione and Wolfenstein-Todel, 1972) it became apparent that the number and sequences around these bonds differed, and appeared to be unique to, and characteristic of, each class, subclass and type. Based on these differences, a simple method was developed for classifying all the currently known immunoglobulin chains (Frangione et al., 1969a; B. Frangione and E. C. Franklin, 1972, unpublished results). Mild thiol reduction selectively breaks interchain bridges, which can then be labeled with iodo[14C]aceticacid. High-voltage electrophoresis of digested proteins gave autoradiographic patterns which were characteristic for each type of heavy and light chain, and allowed classification by simple inspection, “chemical typing” (Frangione et al., 1969a). However, this method was generally insufficient for studying the molecular defect present in HCD proteins and immunoglobulin molecules with other abnormalities. On the other hand, tryptic fingerprint analysis proved difficult to interpret because of the presence of variable peptides. An alternative approach was developed, taking advantage of the facts that a loop (or intrachain bridge) of about 60 residues is a fundamental pseudosubunit which is repeated six times in the heavy- and light-chain monomer of an IgG molecule and seven times in the heavy- and light-chain monomer of an IgM molecule. One such loop in the L chain, three in the y chain and four in the p chain (Frangione et al., 1971)
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are essentially invariant within each class or subclass. Therefore, studies of interchain bridge peptides (chemical typing) combined with those of intrachain bridges can be extremely powerful tools in analyzing specific positions along the chains. Interchain sequences are recognized by the radioactive label technique and intrachain sequences by ninhydrin staining. The first dimension of the map classifies the immunoglobulin chain type, class, or subclass and/or unusual structure (negative results in this step indicate the absence or sequences responsible for interchain binding and lack of dimerization). The second dimension allows studies of fragments along the chains since each domain contains a group of thiol-containing peptides with a distinct position on the map. Absence of one or more peptides defines rather accurately the nature of the defect. It is worth mentioning the possibility that reduction in 0.005 M dithiothreitol may not always distinguish so clearly between inter and intra bonds when examining abnormal proteins. However, no such difficulties were met with the protein examined here and others currently under study, and detailed chemical analysis, molecular weight determination and amino acid sequence (Franklin and Frangione, 1971; Frangione et al., 1973) further corroborated the usefulness of the method. Studies of H chains possessing deletions in various segments of their chains and selected amino acid sequence on the variations found in the “hinge or interdomain” region (between CHI and cH2) and in the so-called “switch” region (between VH and CHI) should aid in establishing the genetic
FLAVINE-HISTIDINE
PEPTIDES
mechanisms which control the synthesis of immunoglobulin heavy chains. Acknowledgment
I thank Dr. Edward C. Franklin for correcting the manuscript and Ms. Marlene Chavis for secretarial assistance. References Brown, J. R., and Hartley, B. S. (1966), Biochem. J . 101: 214. Cooper, S. M., Franklin, E. C., and Frangione, B. (1972), Science 176,187. Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb, P. D., Rutishauser, H., and Eaxdal, M. J. (1969), Proc. Nat. Acad. Sci. U. S . 63,78. Fett, J. W., Deutsch, H. F., and Smities, 0. (1973), Immunochemistry 10,115.
Frangione, B., and Franklin, E. C. (1972), FEBS (Fed. Eur. Biochem. SOC.) Lett. 20, 321. Frangione, B., Lee, L., Haber, E., and Bloch, K. J. (1973), Proc. Nat. Acad. Sci. U. S . 70,1073. Frangione, B., and Milstein, C. (1969), Nature (London) 224, 597. Frangione, B., Milstein, C., and Franklin, E. C. (1969a), Nature (London)221, 149. Frangione, B., Milstein, C., and Pink, J. R. L. (1969b), Nature (London) 221, 145. Frangione, B., Prelli, F., Mihaesco, C., and Franklin, E. C. (1971), Proc. Nat. Acad. Sci. U. S . 68,1547. Frangione, B., and Wolfenstein-Todel, C. (1972), Proc. Nat. Acad. Sci. U . S . 69,3673. Franklin, E. C., and Frangione, B. (1971), Proc. Nat. Acad. Sci. U . S . 68, 187. Prahl, J. W. (1967), Biochem. J . 105,1019. Wolfenstein, C., Frangione, B., and Franklin, E. C. (1971), Biochemistry I O , 4140.
Syntheses and Properties of Flavine-Histidine Peptidest Paul G. Johnson and Donald B. McCormick*
ABSTRACT: Flavinylhistidine peptides were synthesized by attaching the amino group of the histidine moiety through amide linkage to aliphatic carboxylic acid chains of varying length at the N-10 position of the isoalloxazine ring. The fluorescence properties of these models were measured in order to understand more fully flavine-histidine interactions, which can occur in certain flavoprotein systems. Measurements of fluorescence quenching indicate interaction between the isoalloxazine and nonprotonated imidazole ring of the histidine moiety. Very little interaction apparently occurs between flavine and protonated histidine. The effects seen when temperature and solvent are varied seem to indicate that there
I
nteractions of flavine coenzymes and aromatic molecules have been recognized for quite some time (Weber, 1950; Yagi et al., 1959; Strittmatter, 1961 ; McCormick et al., 1967). Recently, this has led to a study of the degree and type of molecular interactions of flavins with such aromatic amino acids as tryptophan, tyrosine, and phenylalanine. Flavine peptide models were synthesized by attaching the amino acid through amide linkage to the N-10 position of the isoalloxazine ring via aliphatic chains of varying length (Fory et al., 1968). A fluorescence spectral investigation of the compounds ? F r o m the Section of Biochemistry, Molecular and Cell Biology and the Graduate School of Nutrition, Cornel1 University, Ithaca, New York 14850. Received April 24, 1973. This work was supported in part by Research Grant AM-04585 from the National Institute of Arthritis and Metabolic Diseases, U. S. Public Health Service, and in part by funds made available through the State University of New York. A preliminary account of this work was presented at the 164th National Meeting of the American Chemical Society, New York, N. Y., Sept 1972.
is greater ground-state association for the short-chain peptides, in which the histidine and flavine intramolecularly interact to form a dark, nonfluorescent complex. There is less complexing apparent in the case of the long-chain peptides and more fluorescence quenching due to increased collisions between the light-excited flavine and the histidine. Proton magnetic resonance measurements of these models in D 2 0 (37") at various pD values suggest a weak intramolecular interaction between the imidazole ring of the histidine moiety and the benzenoid portion of the flavine. Such conformations are disrupted at higher temperatures.
showed that hydrophobic interactions probably cause complex formation in aqueous solution, while a dipole-dipole type of interaction is responsible for intramolecular association of some of the amino acid residues with the flavine portion in nonaqueous media (MacKenzie et a[., 1969). The spatial aspects of the intramolecular interactions of these flavine amino acids, both in aqueous and nonaqueous media, were examined by means of proton magnetic resonance spectroscopy (Fory et al., 1970). The FMN-dependent enzyme, pyridoxine (pyridoxamine) 5 '-phosphate oxidase, is another case where molecular association occurs between the flavine coenzyme and an aromatic compound, in this case, a substrate. Synthesis of spectroscopic flavine-Bs models (McCormick and Johnson, 1972) and examination of their fluorescence properties (McCormick, 1972) again suggested a significant interaction between such chromophores . In the last few years, several flavine-containing enzymes have been shown to have a histidine residue at the active site. BIOCHEMISTRY,
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